Bottom Line:
Reverse transcriptase PCR, RNAi knockdown, and KATP blocker profiling showed that the pinacidil DMR is due to the activation of SUR2/Kir6.2 KATP channels in HepG2C3A cells.Kinase inhibition and RNAi knockdown showed that the pinacidil activated KATP channels trigger signaling through Rho kinase and Janus kinase-3, and cause actin remodeling.The results are the first demonstration of a label-free methodology to characterize the composition and signaling of an endogenous ATP-sensitive potassium ion channel.

ABSTRACTCurrent technologies for studying ion channels are fundamentally limited because of their inability to functionally link ion channel activity to cellular pathways. Herein, we report the use of label-free cell phenotypic profiling to decode the composition and signaling of an endogenous ATP-sensitive potassium ion channel (KATP) in HepG2C3A, a hepatocellular carcinoma cell line. Label-free cell phenotypic agonist profiling showed that pinacidil triggered characteristically similar dynamic mass redistribution (DMR) signals in A431, A549, HT29 and HepG2C3A, but not in HepG2 cells. Reverse transcriptase PCR, RNAi knockdown, and KATP blocker profiling showed that the pinacidil DMR is due to the activation of SUR2/Kir6.2 KATP channels in HepG2C3A cells. Kinase inhibition and RNAi knockdown showed that the pinacidil activated KATP channels trigger signaling through Rho kinase and Janus kinase-3, and cause actin remodeling. The results are the first demonstration of a label-free methodology to characterize the composition and signaling of an endogenous ATP-sensitive potassium ion channel.

f2: Agonist activity of two ion channel ligands, NPPB and flufenamic acid at the GPR35 in HT29 cells.(a) The real time DMR dose responses of NPPB. (b) The real DMR amplitudes of NPPB as a function of its dose, in comparison with the DMR of 1 μM zaprinast as a function of NPPB doses. (c) The real time DMR of 4 μM NPPB in the presence of CID2745687 at different doses. (d) The DMR amplitudes of 4 μM NPPB as a function of CID2745687 dose. (e) The real time DMR dose responses of flufenamic acid. (f) The DMR amplitudes of flufenamic acid as a function of its dose, in comparison with the DMR of 1 μM zaprinast as a function of flufenamic acid dose. (g) The real time DMR of 32 μM flufenamic acid in the presence of CID2745687 at different doses. (h) The DMR amplitudes of 32 μM flufenamic acid as a function of CID2745687 dose. (i) The β-arrestin Tango signal as a function of compound dose. For the desensitization, the cells were pretreated with respective ligands for 1 hr before stimulation with zaprinast at the fixed dose (1 μM). For the antagonist blockage, the cells were pretreated with CID2745687 at different doses for 1 hr before stimulation with respective agonist at a fixed dose. Data represents mean ± s.d. (n = 3).

Mentions:
Second, NPPB (5-nitro-2-(3-phenylpropylamino)benzoic acid), niflumic acid, IAA-94 (R(+)-methylindazone) and flufenamic acid shared similar cell phenotypic pharmacology; all were active in HT29 and C3A cells, and to less extent in HepG2 cells. Recently, we had showed that the positive DMR of niflumic acid in HT29 is due to the activation of GPR3534. Consistent with its known agonist activity at the GPR3535, NPPB triggered a dose-dependent DMR in HT29 (Fig. 2a), yielding an apparent logEC50 of −5.95 ± 0.07 (n = 3) (Fig. 2b). It also desensitized the response to 1 μM zaprinast, a known GPR35 agonist, with a logIC50 of −5.79 ± 0.05 (n = 3) (Fig. 2b). The known GPR35 antagonist CID2745687 dose-dependently and completely blocked the DMR of 4 μM NPPB (n = 3) (Fig. 2c), with an apparent logIC50 of −5.51 ± 0.06 (n = 3) (Fig. 2d). Although flufenamic acid was reported to be inactive in human GPR35-β-arrestin-2 interaction assays36, we found that flufenamic acid is a DMR biased partial agonist at the GPR35 (Fig. 2e–h). Flufenamic acid triggered a dose-dependent DMR in HT29 (Fig. 2e), yielding an apparent logEC50 of −5.18 ± 0.03 (n = 3) (Fig. 2f). It also desensitized the cells responding to the second stimulation with 1 μM zaprinast with a logIC50 of −4.95 ± 0.06 (n = 3) (Fig. 2f). CID274568 dose-dependently and partially blocked the DMR of 32 μM flufenamic acid with a logIC50 of −5.28 ± 0.05 (Fig. 2g & h). Using GPR35 Tango β-arrestin gene reporter assay, we found that NPPB triggered a dose response with a logEC50 of −4.55 ± 0.04 and a maximal response that is 45 ± 3% (n = 3) of the full agonist zaprinast, but flufenamic acid was inactive (Fig. 2i). These results suggest that similar to several other GPR35 agonists including benserazide37, tolcapone38 and rosmarinic acid39, flufenamic acid not only activates the endogenous GPR35 in HT29, but also activates an additional unknown receptor. Of note, we did not determine the mechanism accounted for the DMR of IAA-94 in HT29, given that the IAA-94 DMR is much smaller than those induced by GPR35 agonists.

f2: Agonist activity of two ion channel ligands, NPPB and flufenamic acid at the GPR35 in HT29 cells.(a) The real time DMR dose responses of NPPB. (b) The real DMR amplitudes of NPPB as a function of its dose, in comparison with the DMR of 1 μM zaprinast as a function of NPPB doses. (c) The real time DMR of 4 μM NPPB in the presence of CID2745687 at different doses. (d) The DMR amplitudes of 4 μM NPPB as a function of CID2745687 dose. (e) The real time DMR dose responses of flufenamic acid. (f) The DMR amplitudes of flufenamic acid as a function of its dose, in comparison with the DMR of 1 μM zaprinast as a function of flufenamic acid dose. (g) The real time DMR of 32 μM flufenamic acid in the presence of CID2745687 at different doses. (h) The DMR amplitudes of 32 μM flufenamic acid as a function of CID2745687 dose. (i) The β-arrestin Tango signal as a function of compound dose. For the desensitization, the cells were pretreated with respective ligands for 1 hr before stimulation with zaprinast at the fixed dose (1 μM). For the antagonist blockage, the cells were pretreated with CID2745687 at different doses for 1 hr before stimulation with respective agonist at a fixed dose. Data represents mean ± s.d. (n = 3).

Mentions:
Second, NPPB (5-nitro-2-(3-phenylpropylamino)benzoic acid), niflumic acid, IAA-94 (R(+)-methylindazone) and flufenamic acid shared similar cell phenotypic pharmacology; all were active in HT29 and C3A cells, and to less extent in HepG2 cells. Recently, we had showed that the positive DMR of niflumic acid in HT29 is due to the activation of GPR3534. Consistent with its known agonist activity at the GPR3535, NPPB triggered a dose-dependent DMR in HT29 (Fig. 2a), yielding an apparent logEC50 of −5.95 ± 0.07 (n = 3) (Fig. 2b). It also desensitized the response to 1 μM zaprinast, a known GPR35 agonist, with a logIC50 of −5.79 ± 0.05 (n = 3) (Fig. 2b). The known GPR35 antagonist CID2745687 dose-dependently and completely blocked the DMR of 4 μM NPPB (n = 3) (Fig. 2c), with an apparent logIC50 of −5.51 ± 0.06 (n = 3) (Fig. 2d). Although flufenamic acid was reported to be inactive in human GPR35-β-arrestin-2 interaction assays36, we found that flufenamic acid is a DMR biased partial agonist at the GPR35 (Fig. 2e–h). Flufenamic acid triggered a dose-dependent DMR in HT29 (Fig. 2e), yielding an apparent logEC50 of −5.18 ± 0.03 (n = 3) (Fig. 2f). It also desensitized the cells responding to the second stimulation with 1 μM zaprinast with a logIC50 of −4.95 ± 0.06 (n = 3) (Fig. 2f). CID274568 dose-dependently and partially blocked the DMR of 32 μM flufenamic acid with a logIC50 of −5.28 ± 0.05 (Fig. 2g & h). Using GPR35 Tango β-arrestin gene reporter assay, we found that NPPB triggered a dose response with a logEC50 of −4.55 ± 0.04 and a maximal response that is 45 ± 3% (n = 3) of the full agonist zaprinast, but flufenamic acid was inactive (Fig. 2i). These results suggest that similar to several other GPR35 agonists including benserazide37, tolcapone38 and rosmarinic acid39, flufenamic acid not only activates the endogenous GPR35 in HT29, but also activates an additional unknown receptor. Of note, we did not determine the mechanism accounted for the DMR of IAA-94 in HT29, given that the IAA-94 DMR is much smaller than those induced by GPR35 agonists.

Bottom Line:
Reverse transcriptase PCR, RNAi knockdown, and KATP blocker profiling showed that the pinacidil DMR is due to the activation of SUR2/Kir6.2 KATP channels in HepG2C3A cells.Kinase inhibition and RNAi knockdown showed that the pinacidil activated KATP channels trigger signaling through Rho kinase and Janus kinase-3, and cause actin remodeling.The results are the first demonstration of a label-free methodology to characterize the composition and signaling of an endogenous ATP-sensitive potassium ion channel.

ABSTRACTCurrent technologies for studying ion channels are fundamentally limited because of their inability to functionally link ion channel activity to cellular pathways. Herein, we report the use of label-free cell phenotypic profiling to decode the composition and signaling of an endogenous ATP-sensitive potassium ion channel (KATP) in HepG2C3A, a hepatocellular carcinoma cell line. Label-free cell phenotypic agonist profiling showed that pinacidil triggered characteristically similar dynamic mass redistribution (DMR) signals in A431, A549, HT29 and HepG2C3A, but not in HepG2 cells. Reverse transcriptase PCR, RNAi knockdown, and KATP blocker profiling showed that the pinacidil DMR is due to the activation of SUR2/Kir6.2 KATP channels in HepG2C3A cells. Kinase inhibition and RNAi knockdown showed that the pinacidil activated KATP channels trigger signaling through Rho kinase and Janus kinase-3, and cause actin remodeling. The results are the first demonstration of a label-free methodology to characterize the composition and signaling of an endogenous ATP-sensitive potassium ion channel.